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Title: Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies

Abstract

Membrane contact sites between organelles serve as molecular hubs for the exchange of metabolites and signals. In yeast, the Endoplasmic Reticulum – Mitochondrion Encounter Structure (ERMES) tethers these two organelles likely to facilitate the non-vesicular exchange of essential phospholipids. Present in Fungi and Amoebas but not in Metazoans, ERMES is composed of five distinct subunits; among those, Mdm12, Mmm1 and Mdm34 each contain an SMP domain functioning as a lipid transfer module. We previously showed that the SMP domains of Mdm12 and Mmm1 form a hetero-tetramer. Here we describe our strategy to diversify the number of Mdm12/Mmm1 complexes suited for structural studies. We use sequence analysis of orthologues combined to protein engineering of disordered regions to guide the design of protein constructs and expand the repertoire of Mdm12/Mmm1 complexes more likely to crystallize. Using this combinatorial approach we report crystals of Mdm12/Mmm1 ERMES complexes currently diffracting to 4.5 Å resolution and a new structure of Mdm12 solved at 4.1 Å resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal β-strand; it differs from a previously reported homodimeric structure where the N-terminal β strands where swapped to promote dimerization. Based on our electron microscopy data, wemore » propose a refined pseudo-atomic model of the Mdm12/Mmm1 complex that agrees with our crystallographic and small-angle X-ray scattering (SAXS) solution data.« less

Authors:
; ; ; ORCiD logo
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
UNIVERSITY
OSTI Identifier:
1373779
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 488; Journal Issue: 1
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; ERMESMembrane contact sitesMdm12/Mmm1 complex reconstitutionSMP domainCrystal structureSAXS

Citation Formats

AhYoung, Andrew P., Lu, Brian, Cascio, Duilio, and Egea, Pascal F.. Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies. United States: N. p., 2017. Web. doi:10.1016/j.bbrc.2017.05.021.
AhYoung, Andrew P., Lu, Brian, Cascio, Duilio, & Egea, Pascal F.. Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies. United States. doi:10.1016/j.bbrc.2017.05.021.
AhYoung, Andrew P., Lu, Brian, Cascio, Duilio, and Egea, Pascal F.. Thu . "Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies". United States. doi:10.1016/j.bbrc.2017.05.021.
@article{osti_1373779,
title = {Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies},
author = {AhYoung, Andrew P. and Lu, Brian and Cascio, Duilio and Egea, Pascal F.},
abstractNote = {Membrane contact sites between organelles serve as molecular hubs for the exchange of metabolites and signals. In yeast, the Endoplasmic Reticulum – Mitochondrion Encounter Structure (ERMES) tethers these two organelles likely to facilitate the non-vesicular exchange of essential phospholipids. Present in Fungi and Amoebas but not in Metazoans, ERMES is composed of five distinct subunits; among those, Mdm12, Mmm1 and Mdm34 each contain an SMP domain functioning as a lipid transfer module. We previously showed that the SMP domains of Mdm12 and Mmm1 form a hetero-tetramer. Here we describe our strategy to diversify the number of Mdm12/Mmm1 complexes suited for structural studies. We use sequence analysis of orthologues combined to protein engineering of disordered regions to guide the design of protein constructs and expand the repertoire of Mdm12/Mmm1 complexes more likely to crystallize. Using this combinatorial approach we report crystals of Mdm12/Mmm1 ERMES complexes currently diffracting to 4.5 Å resolution and a new structure of Mdm12 solved at 4.1 Å resolution. Our structure reveals a monomeric form of Mdm12 with a conformationally dynamic N-terminal β-strand; it differs from a previously reported homodimeric structure where the N-terminal β strands where swapped to promote dimerization. Based on our electron microscopy data, we propose a refined pseudo-atomic model of the Mdm12/Mmm1 complex that agrees with our crystallographic and small-angle X-ray scattering (SAXS) solution data.},
doi = {10.1016/j.bbrc.2017.05.021},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 488,
place = {United States},
year = {Thu Jun 01 00:00:00 EDT 2017},
month = {Thu Jun 01 00:00:00 EDT 2017}
}